Akt/protein kinase B (PKB) has been shown to be a widely expressed Ser/Thr protein kinase whose persistent activation leads to human oncogenesis. Its role in cancer and chemoresistance is accomplished by the concomitant promotion of cell growth, migration, and angiogenesis as well as the suppression of the apoptotic pathway. There has been significant interest in Akt for its structural and functional properties as well as its implications in the area of cancer therapy.
The Akt family consists of three members, Akt1 (PKBα), Akt2 (PKBβ), and Akt3 (PKBγ); that are structurally very similar (>85% sequence homology). Each isoform consists of an N-terminal pleckstrin homology (PH) domain, a central catalytic domain, and a C-terminal regulatory tail.
Stimuli activating Akt includes molecules that regulate tyrosine kinase activity and G-protein-linked receptors, cAMP/PKA agonists, and phosphatase inhibitors. Direct activation of Akt is mediated by agonist-induced stimulation of phosphoinositide-3 kinase (PI3K), which generates phosphatidylinositol-3,4,5-triphosphate (PIP3), a lipid second messenger which binds to the PH domain of Akt and translocates it to the intracellular side of the plasma membrane. Anchored to the plasma membrane, Akt then undergoes dual phosphorylation by membrane associated protein kinases PDK1 and PDK2 on a pair of serine and threonine residues respectively (Thr308 in the activation loop and Ser473 in the C-terminal hydrophobic motif). This dual phosphorylation induces a conformational change in the enzyme to its activated form, which incorporates and ATP binding site as well as a substrate binding site.
Akt directly phosphorylates substrates that are involved in the regulation of numerous cellular functions such as cellular proliferation, transcription, migration, apoptosis, cellular differentiation, and metabolism. The disregulation of Akt kinase activity has been detected in a number of human malignancies including ovarian, breast, thyroid, and colon cancers. Amplification or overexpression of Akt results in the up-regulation of cell growth and the down-regulation of apoptosis. The cellular levels of PIP3 regulate the activity of PDK-1, which is responsible for Akt activation. The levels of these phosphoinositides are dependent on the activity of PI3K and phosphatases PTEN and SHIP. Tumor suppressor PTEN negatively regulates the activity of Akt by converting PIP3 back to PIP2.
Inhibition of Akt activity has been shown to suppress cell growth and induce apoptosis in tumor cell lines derived from various organs possessing constitutively activated Akt. Akt activation causes the phosphorylation and inactivation of key cell maintenance proteins, like glycogen synthase kinase-3 (GSK-3). Normally active, Akt phosphorylates Ser21 on GSK-3α or Ser9 on GSK-3β, thereby inactivating GSK-3. GSK-3 is a cytoplasmic serine-theronine kinase existing in two isoforms, GSK-3α (51 kDa) and GSK-3β (47 kDa). The isoforms retain 98% homology in kinase domains, but only 36% homology in the last 76 amino acid residues in the C-terminus. GSK-3 is responsible for regulating cellular metabolism and is involved in insulin, Wnt, developmental and sonic hedgehog signaling pathways.
The majority of small molecule kinase inhibitors target the ATP binding pocket and there have been few reports targeting the substrate binding site. ATP mimetics have met with much success, however selective binding within this pocket remains challenging as these inhibitors compete with the many ATP utilizing enzyme possessing similar contact regions as well as with high cellular concentrations of ATP. Substrate-mimetics offer a promising method for the design of selective in vivo inhibitors of Akt as they can exploit sequence specificity. The substrate binding region has evolved to recognize specific substrate sequences and therefore offers a larger number of potential interactions for a properly designed inhibitor than the corresponding ATP pocket. The inherent design challenges present in substrate-mimetics are the large binding pocket and extended binding conformation of many natural substrates. We recently described the development of substrate-mimetic inhibitors of Akt based on the consensus sequence and the structure of an enzyme bound substrate. The preliminary studies demonstrate that limited structural modification of the initial peptidic substrate can overcome these challenges and provide peptidomimetic inhibitors with increasing lipophilicity, rigidity, and potency as well as decreasing the size and peptidic nature of the inhibitors.